The present invention relates to the field of determining components of biological fluids, and more particularly relates to methods, reagents, test cartridges and devices useful for determining fibrinogen in physiological samples, preferably in undiluted whole blood and undiluted blood plasma samples.
The recognition of a potential role of fibrinogen in cardiovascular disorders has increased the need for reliable and simple fibrinogen assays. Prior art methods of determining fibrinogen include clotting-time dependent fibrinogen assays that determine the coagulation time of a diluted sample and correlate the clotting time with the fibrinogen concentration in a sample wherein the coagulation time is inversely proportional to the amount of fibrinogen. In addition, total protein assays have been performed to determine the amount of protein in a clot, which is then correlated with an initial fibrinogen concentration. The first type of technique requires dilution in order to slow down the rate of coagulation to facilitate obtaining a meaningful result, while the second type of technique requires isolation of a clot from the sample, washing the clot, and determining the protein content. In view of the desire to minimize the manipulation of biological fluids, such as blood, there is a need for improved assay methods that minimize sample manipulation. Thus, it is particularly desirable to have an assay that can utilize an undiluted sample, but which avoids the problems caused by rapid clotting.
The need for improved assay methods that minimize sample manipulation is demonstrated by U.S. Pat. No. 5,292,664, to Fickenscher, which discloses a test and reagent for determining fibrinogen in undiluted plasma samples. The method of Fickenscher involves the addition of a large excess of thrombin, or a protease with analogous activity, to ensure immediate conversion of all of the fibrinogen in the sample to fibrin monomers. In a preferred embodiment of Fickensher""s method, a heparin inhibitor is added to prevent inhibition of thrombin. However, the aggregation of the fibrin monomers is slowed by the addition of a fibrin aggregation inhibitor, thus slowing the formation of clots. The clotting time at a constant concentration of a fibrin aggregation inhibitor can be correlated with the fibrin concentration, and hence lead to a determination of the fibrinogen level in the original sample. Thus, Fickensher""s method of inhibiting clotting promotes a first reaction in the clotting pathway, followed by inhibition of a subsequent reaction. This method requires that the sample be combined with a fibrin aggregation inhibitor prior to the addition of excess thrombin, otherwise clotting will occur too rapidly for the method to be useful.
Further information on the physiological role of fibrinogen and prior assays is abundant and readily available and known to those of skill in the art. More background information can be obtained from: National Committee for Clinical Laboratory Standards, Procedure for the Determination of Fibrinogen in Plasma; Approved Guideline. NCCLS document H30-A. (ISBN 1-56238-221-7), NCCLS, 771 East Lancaster Avenue, Villanova, Penn. 19085 (1994); U.S. Pat. No. 5,563,041; and Canadian Patent 2,252,983. All documents mentioned herein are incorporated by reference as if reproduced in full below.
There remains a need for a one-step fibrinogen assay for whole undiluted blood, and corresponding reagents and devices. The term xe2x80x9cone-stepxe2x80x9d refers to test sample manipulation steps, such as the number of manipulations required to determine the fibrinogen in a sample. There is also a need for clotting-time dependent fibrinogen assays in which sample clotting time is less than about three hundred seconds for samples containing a wide range of fibrinogen concentrations and in which clinically significant differences in sample fibrinogen concentration correlate to reliable and readily measurable differences in clotting time. There is also a need for fibrinogen assay reagents which are stable, cost effective, readily available, can be used in dry form and can be dissolved by the sample of interest, and that provide consistent results from sample to sample. It is also desired that sample containers and other surfaces that come into contact with samples, particularly blood samples, be disposable and provide minimum opportunity for human contact with the sample. Thus, there is also a need for a cartridge useful in an automated clotting time determination device, which fulfils all or most of these needs. There is also a need for an automated device that directly reports the amount of fibrinogen in a sample.
The present invention involves a method, reagent, test cartridge, and device for determining fibrinogen in a physiological sample, such as whole blood or undiluted blood plasma. The method, reagent, test cartridge and device make use of thrombin and a thrombin inhibitor. With the thrombin and thrombin inhibitor added to it, the clotting time of a sample is measured. Since the clotting time of a blood or blood plasma sample is inversely related to its fibrinogen concentration, the clotting time can be used to determine sample fibrinogen concentration by reference to the clotting times of fibrinogen standards that have been contacted with constant amounts of reagent active ingredients.
In a preferred embodiment of the present invention, the physiological sample is undiluted, such as an undiluted whole blood sample or an undiluted blood plasma sample. Preferably, activity of added thrombin, as well as the activity of the endogenous thrombin in blood samples, is inhibited in the assay, thus slowing the rate of conversion of fibrinogen in the sample to fibrin. In a preferred embodiment, clotting time is sufficiently slow in samples containing the range of fibrinogen concentrations of clinical interest that differences in clotting time can be readily linked to different fibrinogen levels. In a preferred embodiment, an assay reagent is utilized that comprises a predetermined amount of thrombin and a thrombin inhibitor that slows but does not stop thrombin enzymatic activity with respect to the conversion of fibrinogen to fibrin.
In a preferred embodiment, a device for determining sample fibrinogen concentration is provided, comprising a cartridge incorporating a reagent of the present invention, comprising thrombin and a thrombin inhibitor. In an embodiment, a test cartridge and reagent is provided which can be utilized in a one-step assay to determine fibrinogen in an undiluted whole blood sample.
In another embodiment, a test cartridge and reagent is utilized in a two-step assay to determine fibrinogen in undiluted blood plasma; the first step being the addition of a detectable material, such as but not limited to latex particles, as a surrogate for red blood cells that would be detected in a whole blood sample, and the second step being identical to the one-step assay for determining fibrinogen in whole blood.
In a preferred embodiment, the device of the present invention comprises a processor for converting sample clotting time to the fibrinogen concentration of the sample. The device preferably includes a display or other readout device for automatically reporting the sample fibrinogen concentration to the device operator, and optionally may also provide the clotting time of the sample. In an alternative embodiment, a reagent and method is provided which can be utilized in a one-step assay using a fibrometer to determine fibrinogen in an undiluted blood plasma sample or undiluted whole blood sample, wherein the clotting time of a sample is based on the time required, after contact of the sample with the reagent, for the fibrometer probe to detect a predetermined degree of resistance to movement through the sample. The clotting time is then used to determine the amount of fibrinogen in the sample.
Thus, one embodiment of the present invention is a device for use in determining fibrinogen in a sample suspected of containing fibrinogen, comprising a vessel for contacting a sample suspected of containing fibrinogen with a reagent that causes conversion of fibrinogen to fibrin thereby causing clotting, a sensor for detecting a predetermined degree of clotting, a timer for determining the amount of time for a predetermined degree of clotting to be reached, a processor that converts clotting time to the amount of fibrinogen in a sample, and a display that can report the amount of fibrinogen in a sample. Preferably, the device further comprises a reagent that causes conversion of fibrinogen to fibrin, wherein the reagent comprises thrombin and a thrombin inhibitor. The display may be electronic or may be a printer that prints out results. The display may also report clotting time, and other parameters.
Preferred thrombin inhibitors (also referred to herein as thrombin activity modulators) for use in the invention include a sulfated polysaccharide, for example dextran sulfate; a peptide with suitable thrombin inhibiting properties, for example Gly-Pro-Ala or Gly-Gly-Arg; a xanthine, for example, caffeine; a polyanetholsulfate; a polyvinylsulfate; bis-lactobionic acid (e.g., aprosulate); a benzamidine, for example a cyclic amide of 4-amidinophenylalanine, such as Nxcex1-(xcex2-naphthylsulfonylglycyl)-4-amidinophenylalanine piperidide; a serine protease inhibitor such as antipain; hirudin, hirulog, bivalirudin, and argatroban. Analogs of these preferred inhibitors that perform in accordance with the present invention are also included herein.
A preferred reagent utilizes a thrombin inhibitor and thrombin at pre-optimized concentrations that can be used to determine fibrinogen in samples containing fibrinogen in concentrations found over the normal physiological range of clinical interest. Preferably, the reagent can be used to reliably determine fibrinogen concentrations in samples having between about 100 mg/dL to about 400 mg/dL fibrinogen. As used herein, the term xe2x80x9cnormal rangexe2x80x9d fibrinogen concentration refers to fibrinogen concentrations between about 100 mg/dL and about 400 mg/dL fibrinogen, while xe2x80x9clow rangexe2x80x9d refers to fibrinogen concentrations between about 0.5 mg/dL and about 100 mg/dL fibrinogen, and xe2x80x9chigh rangexe2x80x9d refers to about 400 mg/dL to about 2,000 mg/dL fibrinogen. In an embodiment, fibrinogen concentrations ranging from about 0.5 mg/dL to about 2000 mg/dL may be determined with the assay, reagent, test cartridge and device of the present invention. Samples containing low range and high range fibrinogen concentrations can be determined by optimizing assay conditions therefore.
In another aspect, the present invention includes a novel cartridge device for use in a clotting time determination device, preferably having the capability of automatically converting the clotting time of a sample to the sample""s fibrinogen concentration, as well as an indicator, electronic display or other read-out device to provide the fibrinogen concentration. Optionally, the device may provide clotting time in addition to fibrinogen concentration, thus, providing a multifunctional device that may be used in other clotting time assays.
In an embodiment, the cartridge includes a housing containing an inlet port, a chamber unit, and an exit port. The cartridge preferably further comprises a first capillary unit for independently pumping (i.e., carrying or transporting) a liquid, for example, an undiluted blood sample, from the inlet port to the chamber unit. In addition, the preferred cartridge preferably includes a second capillary unit positioned between and operatively connected to the chamber unit and the exit port for independently pumping a liquid from the chamber unit to the exit port. The inlet port, first capillary unit (if present), chamber unit, second capillary unit (if present), and exit port are present in a continuous capillary pathway.
In one embodiment, contained within the capillary pathway is a reagent comprising thrombin and a thrombin inhibitor suitable for use in the assay of the present invention. The thrombin and thrombin inhibitor reagent may be formulated in a HEPES buffer matrix. A stabilizer, such as albumin, may also be present in the reagent. In another embodiment, the cartridge contains thrombin reagent, and is utilized in the assay of the present invention by passing through the cartridge a sample containing fibrinogen and a suitable thrombin inhibitor. In an alternative embodiment, the reagent in the cartridge further comprises blood cell surrogates, for example latex particles, in the reagent chamber that mix with the sample, preferably a sample such as blood plasma or synthetic blood lacking more readily detectable particulates.
In another alternative embodiment, the reagents and assay of the present invention can be used with traditional wet chemical techniques. For example, in an embodiment, a fibrometer is used to measure clotting time of fibrinogen containing samples mixed with solubilized reagents of the present invention.